Energy Sensing and Metabolism in Cancer

Transcription

1 Energy Sensing and Metabolism in Cancer Boyi Gan MD Anderson Cancer Center

2 The distinguishing features of living organisms 1. A high degree of chemical complexity and microscopic organization. 2. Systems for extracting, transforming, and using energy from the environment. 3. Defined functions for each of an organism s components and regulated interactions among them. 4. Mechanisms for sensing and responding to alterations in their surroundings. 5. A capacity for precise self-replication and selfassembly. (Lehninger Principles of Biochemistry, 6 th edition)

3 ATP: the molecular unit of currency of intracellular energy transfer Biosynthesis Muscle contraction Membrane transport 1.How cells sense and respond energy and nutrient availability? 2.How cancer cells adapt to survive and grow under metabolic stress? Cellular movement

4 heat Energy Intake Energy Balance Energy Expenditure Obesity Diabetes Aging Cancer

5 Energy Metabolism and Cancer Development Obesity is the second greatest risk factor for cancer development in USA (only after tobacco use). Deregulated energy metabolism is an emerging hallmark of cancer. Our understanding of energy metabolism in cancer has been translated into cancer detection (FDG-PET) and treatment (metformin, the widely used drug to treat diabetes, is associated with decreased tumor incidence and mortality. (Molecular Biology of the Cell, 6 th edition; Hallmarks of cancer: the next generation. Hanahan, Weinberg, Cell, 2011; The biology of cancer, 2 nd edition, Weinberg, 2014)

6 Research Topic: Energy Sensing and Metabolism Research Questions: Cancer 1.How normal/cancer cells sense energy availability? 2.How cancer cells adapt to survive and grow under energy stress? 3.How to translate our understanding of energy metabolism in cancer into novel cancer therapeutics? Presentation Outline: Regulation of energy sensor AMPK by lncrna NBR2. Energy stress-induced lncrna FLINC1 regulates energy metabolism and tumor suppression. Glutamate/cystine antiporter SLC7A11 regulates glucose dependency in cancer cells.

7 AMPK-mediated Energy Sensing Signaling Energy Stress Tuberous Sclerosis, LAM, kidney and bladder cancer AMP/ATP AMPKα β γ LKB1 Peutz Jeghers Syndrome, non-small cell lung cancer, pancreatic cancers, melanomas, cervical cancer ATP-generating TSC1-TSC2 catabolic processes ATP-consuming anabolic processes Restoring mtorc1 energy balance Protein Synthesis, Cell Growth/Size Increase Tumor Suppression Stem Cell Homeostasis

8 AMPK-mediated Energy Sensing Signaling Energy Stress NBR2 (Liu et al, 2006, NCB) AMP/ATP AMPKα β γ LKB1 LKB1 is required for HSC maintenance in vivo (Gan et al, 2010, Nature) TSC is required for HSC maintenance in vivo (Gan et al, 2008, PNAS) TSC1-TSC2 mtorc1 Protein Synthesis, Cell Growth/Size Increase Tumor Suppression Stem Cell Homeostasis

9 Glucose starvation induces lncrna NBR2 expression Genomic duplication and alteration 218 bp NBR2 (neighbor of BRCA1 gene 2) for further study BRCA1: protein-coding gene, regulate DNA damage response and genome integrity NBR2: long non-coding RNA, non-coding gene BRCA1P1: BRCA1 pseudogene, non-coding gene NBR1: protein-coding gene, function as autophagy receptor

10 LKB1-AMPK-dependent induction of NBR2 by energy stress A C Lkb1 deficient B

11 NBR2 depletion attenuates energy stress-induced AMPK activation and mtorc1 inactivation Ctrl Glucose sh sh1 sh2 (mm): p-ampk AMPK p-acc ACC p-raptor Raptor p-s6k S6K p-s6 S6 NBR2 regulates cell proliferation, apoptosis, and autophagy downstream of AMPK under energy stress. Overexpression of NBR2 activates AMPK

12 NBR2 interacts with AMPKα Glucose (mm): AMPKα pampk AMPKβ β1 β2 AMPKγ1 (Using biotinylated RNA, precipitated with streptavidin beads)

13 NBR2 promotes AMPK kinase activity

14 NBR2: A lncrna switch for AMPK activation A NBR2 B (Liu X, et al, Gan B, Nature Cell Biology, 2016)

15 Biguanides (Metformin/Phenformin) as anti-cancer drugs Metformin was originally developed from natural compounds found in the plant known as French lilac. Biguanides (Metformin/Phenformin) are inhibitors of mitochondrial respiratory chain complex I, and can decrease blood glucose levels. Metformin is most widely used drug to treat diabetes. Retrospective analyses, clinical trials and many functional studies support the beneficial effect of biguanides in cancer prevention and treatment. At least three mechanisms have been proposed to explain the antitumor effects of biguanides. (from Hardie DG, 2013, Cancer Cell)

16 Biguanides induce NBR2 expression and NBR2 regulates cancer cell sensitivity to biguanides A Relative NBR2 levels * 16 Control 14 ** Phenformin ** ** 6 ** SLR20 BT549 HEK-293T 786-O MDA-MB-231 B Apoptosis(%) Control Ctrl sh NBR2 sh1 NBR2 sh2 *** Phenformin Phenformin Cleaved PARP -Actin Ctrl sh NBR2 sh1 NBR2 sh

17 NBR2 does not regulate biguanide-induced AMPK activation A B

18 NBR2 regulates biguanide-induced GLUT1 expression and glucose uptake A B

19 Differential effects of NBR2 under glucose starvation and biguanide treatment Glucose Starvation LKB1-AMPK Biguanide treatment? NBR2 NBR2? AMPK GLUT1 mtorc1 activation, Cell Proliferation, Tumor Development Glucose uptake, Cancer cell survival (Liu X, et al, Gan B, Nature Cell Biology, 2016) (Liu X, Gan B, Cell Cycle, 2016)

20 Metabolic Stress TFs (metabolic stress response) RNA sequencing Computational Analysis TCGA Analysis lncrnas Function Validation Metabolism Molecular Mechanism Clinic

21 FoxO transcription factors: at the crossroad of cancer and metabolism (From Salih DA, Brunet A, Curr Opin Cell Biol. 2008) LncRNA Our previous studies showed FoxOs play critical roles in mediating energy stress response, drug resistance, and tumor suppression in renal cancer (Gan B, et al, Cancer Cell, 2010; Lin A, et al, Gan B, Oncogene, 2013; Lin, et al, Gan B, Cancer Research, 2014; Dai F, et al, Gan B, PNAS, 2017).

22 Identification of FoxO-induced lincrnas FoxO(TA)ERT2 RCC cells 4OHT 12 hr treatment - + RNA sequencing Analysis focusing on lincrnas FILNC1 TCGA datasets FILNC1 is a FoxO target. FILNC1 is induced by energy stress in a FoxO-dependent manner FILNC1 (FoxO-induced long noncoding RNA #1)

23 FILNC1 deficiency inhibits energy stress-induced apoptosis and promotes renal tumor development A B 1% Glucose medium shluc sh1 sh2 C FILNC1 KD does not affect cell cycle.

24 FILNC1 is down-regulated and its low expression correlates with poor clinic outcome in renal cancers A B C TCGA analysis Real-time PCR validation Clinical outcome analysis

25 FILNC1 deficiency promotes glucose uptake and lactate production

26 FILNC1 deficiency increases Myc protein level FILNC1 KD does not affect Myc mrna level. Myc largely mediates the biological effects afforded by FILNC1 deficiency under energy stress

27 FILNC1 interacts with AUF1 under energy stress and sequesters AUF1 from binding to Myc mrna Mass spectrometry for FILNC1 Beads Antisense Sense IGF2BP AUF SART AUF1 is an (A+U)-rich elements (AREs)-binding protein, and can binds to AREs within 3 untranslated region (UTR) of Myc mrna and promotes Myc translation without affecting Myc mrna level. B C

28 Energy stress-induced lncrna FILNC1 inhibits Mycmediated energy metabolism and renal tumor suppression (Xiao Z, et al, Gan B, Nature Communications, 2017)

29 Potential function of LncRNAs in cancer metabolism

30 Energy Stress (Short-term and mild stress) AMP/ATP (Long-term and severe stress) Adaptive Response (AMPK activation) Suicidal Response (FoxO activation) Cell Death (Gan et al, Cancer Cell, 2010; Lin et al, Oncogene, 2013; Lin et al, Cancer Research, 2014; Liu X, et al, Nature Cell Biology, 2016; Dai et al, PNAS, 2017; Xiao Z, et al, Nature Communications, 2017)

31 Glucose starvation induces the expression of SLC7A11 0h 4h 8h (-Glc) SLC7A11 * * ** -Glc (h) SLC7A11 Vinculin -Glc (h) SLC7A11 Vinculin P-AMPK NCI-H226 RCC4 BJ SLR20 U2OS AMPK UMRC6 786-O UMRC-6 31

32 Glucose (Glc) Glutathione SLC7A11 (xct) (catalytic subunit) SLC3A2 (regulatory subunit) Cysteine System X c Cystine Cystine Glycolysis ROS (Glu) Pyruvate (Pyr) Pyr TCA Cycle α ketoglutarate (α KG) (Glu) Glutamate (Glu) Glutamine (Gln) 32

33 Hypothesis: glucose starvation induced SLC7A11 serves as an adaptive response to promote survival under metabolic stress Glucose starvation (Adaptive response) SLC7A11 H2DCFDA (ROS) +Glc +Glc, NAC Glc Glc, NAC NAC ROS GSH Con NAC Cell death +Glc Glc UMRC6 (NAC: N acetylcysteine) 33

34 High SLC7A11 expression correlates with increased sensitivity to glucose starvation induced cell death in cancer cells SLC7A11 Vinculin SLC7A11 high SLC7A11 low 34

35 SLC7A11 sensitizes cancer cell to glucose starvation induced cell death shcon shslc-1 shslc-2 EV SLC7A11 +Glc +Glc -Glc -Glc SLC7A11 Vinculin shcon sh SLC-1 sh SLC-2 UMRC6 ** *** SLC7A11 Vinculin EV OE 786-O SLC7A11 Vinculin shcon sh SLC-1 sh SLC-2 NCI-H226 ** *** SLC7A11 Vinculin EV OE RCC4 Cell Death (PI Staining, %) RCC4 EV SLC7A11-WT **** 35 Pharmacological inhibition of SLC7A11 by sulfasalazine has similar effect Glc (h)

36 ATF4 and NRF2 promotes glucose starvation induced cell death through SLC7A11 Glc (+) (-) sgcon + Glc - Glc sgnrf2-1 sgcon sgnrf2-1 sgnrf2-2 sgcon sgnrf2-1 sgnrf2-2 SLC7A11 sgnrf2-2 NRF2 Vinculin UMRC6 Glc (+) (-) + Glc - Glc sgcon sgcon sgatf4-1 sgatf4-2 sgcon sgatf4-1 sgatf4-2 SLC7A11 sgatf4-1 ATF4 Vinculin sgatf4-2 UMRC6 36

37 SLC7A11 regulation of glutamate efflux underlies SLC7A11 mediated increased sensitivity to glucose starvation *** **** **** **** **** *** AOA, EGCG **** *** Inhibition of glutamate conversion to akg by AOA or EGCG restores glucose dependency in SLC7A11-deficient cells. 37

38 Model High glucose / Low SLC7A11 Glucose Glutamine Low glucose / Low SLC7A11 Glucose Glutamine TCA Cycle Glutamate TCA Cycle Glutamate SLC7A11 Glutamate Cystine SLC7A11 Glutamate Cystine High glucose / High SLC7A11 Glucose Glutamine Low glucose / High SLC7A11 Glucose Glutamine TCA Cycle Glutamate TCA Cycle Glutamate SLC7A11 Cystine SLC7A11 Cystine Glutamate Glutamate SLC7A11 limits metabolic flexibility and enhances cancer cell dependency on glucose by exporting glutamate. Suggest to use glycolysis inhibitors to target the metabolic vulnerability in tumors with high SLC7A11 expression (such as Keap1 or NRF2 mutant tumors). (Koppula P, Zhang Y, et al, Gan B, 2017, JBC) 38

39 Research Topic: Energy Sensing and Metabolism Cancer Research Questions: 1.How normal/cancer cells sense energy availability? 2.How cancer cells adapt to survive and grow under energy stress? 3.How to translate our understanding of energy metabolism in cancer into novel cancer therapeutics? Presentation Outline: Regulation of energy sensor AMPK by lncrna NBR2. (Liu X, et al, NCB, 2016; Liu X, et al, Cell Cycle, 2016) Energy stress-induced lncrna FLINC1 regulates energy metabolism and tumor suppression. (Xiao Z, et al, Nature Communications, 2017) Glutamate/cystine antiporter SLC7A11 regulates glucose dependency in cancer cells. (Koppula, JBC, 2017)

40 Acknowledgement Current Lab Members: Li Zhuang Hyemin Lee ZhenDong Xiao Xiaowen Liu Yilei Zhang RajKumar Yadav Anoop chauhan Pranovi Koppula Collaborators: Xifeng Wu Christopher Wood Junjie Chen Han Liang Hui-kuan Lin Deepak Nagrath Wei Li Funding: